{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T21:58:42Z","timestamp":1761947922481},"reference-count":31,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microb Cell Fact"],"published-print":{"date-parts":[[2013,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>Failure of colony PCRs in green microalga <jats:italic>Chlorella vulgaris<\/jats:italic> is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in <jats:italic>C. vulgaris<\/jats:italic> presents a challenge.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of <jats:italic>C. vulgaris.<\/jats:italic> The higher lipids accumulated in <jats:italic>C. vulgaris<\/jats:italic> negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in <jats:italic>C. vulgaris<\/jats:italic>. By simply pipetting\/votexing the pellets of <jats:italic>C. vulgaris<\/jats:italic> in 10 ul of either TE (10 mM Tris\/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast <jats:italic>rbcL<\/jats:italic> gene. This colony PCR protocol is effective and robust in <jats:italic>C. vulgaris<\/jats:italic> and also demonstrates its effectiveness in other <jats:italic>Chlorella<\/jats:italic> species.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>The accumulated lipids rather than the rigid cell walls of <jats:italic>C. vulgaris<\/jats:italic> significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in <jats:italic>Chlorella<\/jats:italic>.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1475-2859-12-106","type":"journal-article","created":{"date-parts":[[2013,11,13]],"date-time":"2013-11-13T06:01:56Z","timestamp":1384322516000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris"],"prefix":"10.1186","volume":"12","author":[{"given":"Crystal Jing Ying","family":"Tear","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chanyuen","family":"Lim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinchuan","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hua","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2013,11,13]]},"reference":[{"key":"897_CR1","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1016\/j.rser.2009.07.020","volume":"14","author":"TM Mata","year":"2010","unstructured":"Mata TM, Martins AA, Caetano NS: Microalgae for biodiesel production and other applications: a review. 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